Stem cell enhancing therapeutics

ABSTRACT

The present application disclose a method for treating a patient who would benefit from stimulation of the patient&#39;s stem cells, comprising administering to the patient an antibody that specifically binds to an epitope of the MUC1 protein expressed on human undifferentiated stem cells.

BACKGROUND

A problem encountered by oncologists is that they must balance thebenefit of chemotherapy against the risk of killing the patient duringthe course of killing their cancers. One of the life-threatening sideeffects of chemotherapy is that cytotoxic drugs kill cancer cells butalso kill healthy cells and in particular kill the patients' stem cells.Stem cells in the bone marrow are constantly regenerating to supply thebody with red blood cells, which carry oxygen, white blood cells, whichfight infection and platelets which cause the blood to clot. Chemo andradiation can destroy the stem cells which eventually become blood cellsand thus put the patient's life at risk, which makes the physicianreduce the cancer killing treatments. The problem with designing agentsto counter these deleterious effects of cancer treatment is that bloodcells are terminally differentiated cells—meaning that they cannotdivide to replicate themselves. They developed from hematopoietic stemcells in the bone marrow. This means that it is not possible to collectsome red or white blood cells from a patient and expand them in vitrothen inject them back into the patient.

There are currently a handful of drugs on the market that are used tomodulate stem cell development in a patient. The first group, known aserythropoiesis stimulating agents (ESAs), includes Epoetin, marketedunder the trade names Procrit and Epogen, and Aranesp. These drugs areused to treat anemia in patients with chronic kidney disease and incancer patients with chemotherapy-induced anemia. These drugs do notstimulate the growth of stem cells in the bone marrow but rather skewthe development of the patient's stem cells such that more become bloodcells of the erythroid lineage. Epogen and epogen-like drugs help cancerpatents and chronic kidney disease patients by increasing the number ofstem cells in the bone marrow that differentiate into red blood cells.

Another class of drugs that is used to stimulate the production of whiteblood cells. Colony-stimulating factors (CSFs) that include G-CSF(granulocyte-colony stimulating factors: marketed as Filgrastim) andGM-CSF (granulocyte-macrophage colony stimulating factors: Sargamostimmarketed as Leukine) stimulate the production of the precursors of whiteblood cells, which can mature to become neutrophils, macrophages anddendritic cells and may be administered with or withoutcyclophosphamide. The CSFs are also stem cell mobilizers which causeblood cell progenitors to be secreted from the bone marrow. However, adrawback of G-CSF and GM-CSF is that they inhibit bone formation.Another mobilizer of hematopoietic stem cells is the CXCR4 antagonistAMD3100, which reportedly mobilizes blood cell progenitors withoutinhibiting bone formation.

Both erythropoiesis stimulating agents (ESAs) and CSFs can be used toaccelerate the recovery of blood cells from effects of chemotherapy,used after bone marrow transplant, used before or after stem celltransplant which may be transplanted into the peripheral blood, and orto treat a patient who could benefit from increased production ofhematopoietic stem cells or blood cells or their progenitors.

Both ESAs and CSFs function by skewing the maturation of stem cellstoward the hematopoietic stem cell lineages and necessarily away frommaturation into other types of cells. It follows that there may beunwanted and dangerous side effects stemming from the inhibition ofother types of cells that are produced in the bone marrow. In fact, theFDA recently issued warnings that the use of Procrit, Epogen or Aranespincreases the risk of developing cancers and also increases the risk ofheart attack and stroke.

Thus an improvement would be the development of agents that stimulatethe production of or skew the development of stem cells to blood celllineages via a pathway that is different from that of the ESAs and theCSFs. A greater improvement to the state of the art would be thedevelopment of agents that stimulate the production of stem cells in thebone marrow rather than just skewing their maturation in one direction.An even greater improvement to the state of the art would be thedevelopment of an agent(s) that would stimulate the growth of stem cellsbut would not stimulate the growth of cancer cells or increase thepatient's risk of developing another cancer. A yet greater improvementwould be the development of an anti-cancer agent that would kill thecancer cells without killing the patient's stem cells.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method fortreating a patient whose blood cells or bone marrow cells have beendepleted, which may occur in some instances as a result of exposure toradiation, the method including administering to the patient a stem cellspecific antibody. In another aspect, the present invention is directedto a method for treating a patient suffering from low blood cell countby stimulating the growth of hematopoietic stem cells or progenitorcells by administering to the patient a stem cell specific antibody.

In another aspect, the present invention is directed to a method ofstimulating growth of bone marrow cells, hematopoietic stem orprogenitor cells in vitro and then administering to a patient sufferingfrom low blood cell count in the peripheral blood or in the bone marrow,in which the method includes administering to the patient a stem cellspecific antibody.

In another aspect, the present invention is directed to a method ofdiagnosing cancer by testing a patient sample with an antibody thatpreferentially binds cancer cells compared to its binding to stem cells.

In a preferred embodiment, the cancer specific antibodies of theinvention are administered to a patient whose treatment regime alsoincludes treatment with stem cell specific antibodies.

In another aspect, the present invention is directed to a method ofselecting therapeutic antibodies for the treatment of cancer involvingde-selecting those antibodies that bind to stem cells. Similarly, thepresent invention is directed to a method of selecting therapeuticantibodies for the treatment of conditions requiring regeneration ofblood cells or bone marrow cells, involving de-selecting thoseantibodies that bind to cancer cells.

In one aspect, the present invention is directed to a method fortreating a patient who would benefit from stimulation of the patient'sstem cells, comprising administering to the patient an antibody thatspecifically binds to an epitope of the MUC1 protein expressed on humanundifferentiated stem cells. The antibody specifically may bind to atleast six consecutive amino acids of the peptide of SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11. Inparticular, the antibody may not bind to the peptide of SEQ ID NO:3, SEQID NO:7 or SEQ ID NO:8. The patient may suffer from cancer. The patientmay suffer from cancer and is receiving chemotherapy or radiation. Or,the patient may suffer from chronic kidney disease.

In another aspect, the invention is directed to a method for treating apatient suffering from cancer or at risk of developing cancer,comprising administering to the patient an antibody that specificallybinds to an epitope of the MUC1 protein that is expressed on cancercells but is not expressed on human undifferentiated stem cells. Theantibody may specifically bind to the peptide of SEQ ID NO:3 and may notbind to the peptide of SEQ ID NO:4.

In still another aspect, the invention is directed to a method fortreating a patient diagnosed with a MUC1-positive cancer comprisingadministering to the patient a monovalent cancer cell-specific antibody.

In another aspect, the invention is directed to a method for treating apatient diagnosed with a MUC1-positive cancer comprising administeringto the patient a monovalent cancer cell-specific antibody.

In another aspect, the invention is directed to a method for treating apatient diagnosed with a MUC1-positive cancer comprising administeringto the patient a monovalent cancer cell-specific antibody and bivalentstem cell specific antibody.

In another aspect, the invention is directed to a method forproliferating stem or progenitor cells, comprising contacting the cellswith an antibody that specifically binds to an epitope of the MUC1protein expressed on human stem cells, without causing proliferation ofcancer cells.

In yet another aspect, the invention is directed to a method ofobtaining an antibody that specifically binds to stem cells, but not tocancer cells comprising the steps of:

(i) generating a mixed set of antibodies that recognize a peptide whosesequence is that of any of the peptides having sequence of SEQ IDNOS:1-11;

(ii) selecting those antibodies that bind to a peptide of SEQ ID NO:4,SEQ ID NO:5, or SEQ ID NO:6, but not to the peptide of SEQ ID NO:3;

(iii) selecting those antibodies that when adsorbed onto a surfacefacilitate the attachment of stem cells; and

(iv) selecting those antibodies that in the bivalent form stimulate thegrowth of stem cells and in the monovalent form inhibit the growth ofstem cells, while having no effect on cancer cells.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cartoon depicting how bivalent antibodies that recognizedMUC1* receptor stimulate growth, but monovalent Fabs of those antibodiesinhibit growth by blocking its interaction with its cognate ligand,dimeric NM23. Bivalent antibodies mimic activity of ligand NM23 to makecancer cells or stem cells grow; monovalent Fab blocks their growth.

FIG. 2 is a graph from an ELISA experiment showing binding of variousantibody clones to either a PSMGFR peptide lacking the N-terminal 10amino acids (N delta 10: SEQ ID NO:3) or a PSMGFR peptide lacking theC-terminal 10 amino acids (C delta 10: SEQ ID NO:4). FIG. 2 shows titerof subclones on MUC1* and MUC1* on peptides SED ID NO:3, “N delta 10”and SEQ ID NO:4, “C delta 10”.

FIG. 3 shows FACS showing the response of MUC1-positive T47D breastcancer cells to either cancer cell specific antibody C2 (MIN-C2) or stemcell specific antibody C3 (2D6C3) plus the data presented graphically.FIG. 3 shows FACS summary of live MUC1* (+) breast cancer cells (T47Dcells)—only cancer cell antibodies (C2) recognize cancer cells; stemcell antibodies (C3) do not.

FIG. 4 shows FACS showing the response of MUC1-positive ZR-75-1 breastcancer cells to either cancer specific antibody C2 (MIN-C2) or stem cellspecific antibody C3 (2D6C3) plus the data presented graphically. FIG. 4shows FACS summary of live MUC1* (+) breast cancer cells (1500 (ZR-75-1cells)); monoclonal antibody C2 (MIN-C2) binds to cancer cells. C3(2D6C3) does not bind to breast cancer cells.

FIG. 5 shows FACS showing the response of MUC1-positive DU145 prostatecancer cells to either cancer specific antibody C2 (MIN-C2) or stem cellspecific antibody C3 (2D6C3) plus the data presented graphically. FIG. 5shows FACS summary of MUC1* (+) prostate cancer cells (DU145 cells):cancer cell antibodies C2 (MIN-C2) and E6 (MIN-E6) bind to MUC1-positiveprostate cancer cells DU145; stem cell antibody C3 (2D6C3) does not.

FIG. 6 shows FACS showing the response of either MUC1-positive embryonicBGO1V/hOG human stem cells, MUC1-positive T47D breast cancer cells, orMUC1-positive DU145 prostate cancer cells to stem cell specific antibodyC3 (2D6C3) plus the data presented graphically. FIG. 6 shows that stemcell antibody (C3) recognizes stem cells but not cancer cells.

FIG. 7 shows photographs of BGO1V/hOG embryonic stem cells growing inplates in the presence of either the control 8 nM NM23 (dimeric),200ug/ml bivalent C3 (2D6C3) stem cell specific monoclonal antibody, or200 ug/ml bivalent C8 (2D6C8) stem cell specific monoclonal antibody.Resultant cells were measured after 72 hour growth period and measuredin a Vialight assay and graphed as shown. FIG. 7 shows that bivalentstem cell antibodies C3 (2D6C3) and C8 (2D6C8) stimulate stem cellgrowth better than the natural ligand NM23.

FIG. 8 shows photographs of BGO1V/hOG embryonic stem cells growing inplates in the presence of either the control 8 nM NM23 (dimeric) orbivalent C3 (2D6C3) stem cell specific monoclonal antibody at theconcentrations shown. Resultant cells were measured after 72 hour growthperiod and measured in a Vialight assay and graphed as shown. Cell countwas measured using vialight assay.

FIG. 9 shows photographs of BGO1V/hOG embryonic stem cells growing inplates in the presence of either the control 8 nM NM23 (dimeric) orbivalent C8 (2D6C8) stem cell specific monoclonal antibody at theconcentrations shown. Resultant cells were measured after 72 hour growthperiod and measured in a Vialight assay and graphed as shown. Cell countwas measured using vialight assay.

FIG. 10 shows photographs of BGO1V/hOG embryonic stem cells growing inplates in the presence of either the control 8 nM NM23 (dimeric) or theFab of either the cancer specific monoclonal antibody C2 (MIN-C2) or theFab of stem cell specific monoclonal antibody C3 (2D6C3) at theconcentrations indicated, showing that only the C3 Fab at 100 ug/mlsignificantly inhibited the growth of stem cells.

FIG. 11 shows photographs of BGO1V/hOG embryonic stem cells growing inplates in the presence of either the control 8 nM NM23 (dimeric) or theFab of cancer specific monoclonal antibodies C2 (MIN-C2) and E6(MIN-E6), showing that neither cancer specific antibody inhibited thegrowth of stem cells.

FIG. 12 shows a graph of measured Tra 1-60, a marker on undifferentiatedstem cells, after stem cells were treated with either the control (Ctrl)8 nM NM23 (dimeric) or the Fab of either stem cell specific monoclonalantibodies C3 (2D6C3) and C8 (2D6C8) or with the Fab of cancer specificmonoclonal antibody E6 (MIN-E6). Results show that by another measure,the cancer specific antibody did not inhibit the growth of stem cells.FIG. 12 shows that Fab's of the stem cell antibodies inhibit stem cellgrowth and cause loss of pluripotency marker Tra 1-60; here we show thatthe Fab's of stem cell monoclonal antibodies C3 and C8 decreaseexpression of stem cell marker Tra 1-60; the Fab of cancer cell antibodyE6 has no effect on inhibition of BG01V cell growth: effect of Fab'smeasured at t=72 hrs.

FIG. 13 shows photographs of MUC1-positive DU145 prostate cancer cellsafter treatment with either the Fab of cancer specific antibody C2(CA-C2-Fab (MIN-C2)), Fab of E6 (CA-E6-Fab (MIN-E6)) or the Fabs of stemcell specific antibodies C3 (STEM-C3-Fab (2D6C3)) or C8 (STEM-C8-Fab(2D6C8)), showing that only the Fabs of the cancer specific antibodiesinhibited the growth of cancer cells. FIG. 13 shows that stem cellspecific mAbs (C3 and C8) do not bind to DU145 prostate cancer cells anddo not inhibit cancer cell growth; Fab's of cancer specific mAbs (C2 andE6) do. The Fabs bind to and block ligand-induced dimerization of MUC1*.

FIG. 14 shows photographs and graphs of cell counts of MUC1-positiveT47D breast cancer cells after treatment with either the Fab of cancerspecific antibody C2 (CA-C2-Fab (MIN-C2)), Fab of E6 (CA-E6-Fab(MIN-E6)) or the Fabs of stem cell specific antibodies C3 (STEM-C3-FAB(2D6C3)) or C8 (STEM-C8-Fab (2D6C8)), showing that only the Fabs of thecancer specific antibodies inhibited the growth of cancer cells. FIG. 14shows stem cell specific mAbs (C3 and C8) do not bind to cancer cellsand do not inhibit cancer cell growth; Fabs of cancer specific mAbs (C2and E6) do.

FIG. 15 shows photographs and graphs of cell counts of MUC1-positiveDU145 prostate cancer cells after treatment with either the Fab ofcancer specific antibody C2 (C2-Fab (MIN-C2)), Fab of E6 (E6-Fab(MIN-E6)) or the Fabs of stem cell specific antibodies C3 (C3-Fab(2D6C3)) or C8 (C8-Fab (2D6C8)), showing that only the Fabs of thecancer specific antibodies inhibited the growth of cancer cells.

FIG. 16 is a graph of cell counts of MUC1-negative PC3 prostate cancercells, which are MUC1 negative, after treatment with either the Fab ofcancer specific antibody C2 (C2-Fab (MIN-C2)), Fab of E6 (E6-Fab(MIN-E6)) or the Fabs of stem cell specific antibodies C3 (C3-Fab(2D6C3)) or C8 (C8-Fab (2D6C8)), showing that none of the Fabs had anyeffect on MUC1-negative cancer cells.

FIG. 17 is the graphical representation of the results of an experimentin which nude mice were xenografted with human T47D breast tumors thentreated with either vehicle (control) or 80 mg/kg E6 (MIN-E6) Fab2-times per week. Treatment commenced at Day 14 post implantation, thenwas suspended for 15 days, then resumed. FIG. 17 shows that human breastcancer tumors (T47D) were implanted into female Nu/Nu mice with estrogenpellets; anti-MUC1* Fab inhibited tumor growth.

FIG. 18 shows that anti-MUC1* Fab shrinks tumors and reduces expressionof MUC1* growth factor receptor. FIG. 18 shows photographs of two of themice from that study.

FIG. 19 shows a graph of tumor volume for NOD/SCID mice xenografted withhuman prostate tumor line DU145, wherein half the mice were treated withE6 Fab at 160 mg/kg every 48 hours and the other half treated withvehicle alone. FIG. 19 shows anti-MUC1* Fab inhibits growth of DU145tumors implanted into male NOD/SCID mice. miR-145 except for V8, lowmiR=high tumor volumes, except for Fab 5, high miR-145=low tumor volume.

FIG. 20 shows Western blot and a graph of microRNA 145 levels fromtumors excised from mice of the study, showing that in general, thetumors treated with the E6 Fab changed to have less MUC1* growth factorreceptor than the control group and more microRNA145 which signals cellsto differentiate. FIG. 20 shows that animals treated with anti-MUC1* Fabexpress less MUC1* growth factor receptor post treatment and become moredifferentiated (miR-145 increases).

FIG. 21 shows a graph of tumor volume for NOD/SCID mice xenografted withhuman prostate tumor line DU145, wherein half the mice began treatmentwhen their tumors averaged over 400 mm³ and the other half begantreatment when their tumors were between 175 mm³ and 300 mm³. Half ofthe large tumors were treated with the E6 Fab and the other half thevehicle alone. Similarly, the second group was divided into treated withE6 Fab at 160 mg/kg every 48 hours and the other half treated withvehicle alone. Results show that the Fab reduced tumor growth rate inboth groups, however the group that began with the smaller tumors didmuch better. FIG. 21 shows normalized tumor growth—DU145 human prostatecancer in NOD/SCID male mice. Groups 1 and 2 tumors were very largeafter 27 days growth—350-550 mm³ at start of treatment. Groups 3 and 4tumors were 175-300 mm³ at start of treatment. Despite size difference,both vehicle groups quickly merged to the same growth rate and size;smaller tumors responded better than the very large tumors as expected;160 mg/kg every 48 hrs.

FIG. 22 shows photos of mice bearing human prostate tumors that receivedthe mock treatment.

FIG. 23 shows photos of mice bearing human prostate tumors that receivedthe cancer specific E6 (MIN-E6) Fab.

FIG. 24 shows anti-MUC1* IgG monoclonal antibody light chain variableregion sequences.

FIG. 25 shows anti-MUC1* IgG monoclonal antibody heavy chain variableregion sequences.

FIG. 26 shows the amino acid sequence of a MIN-C2 (single chain fragmentvariable) design (heavy chain variable—linker—light chain variable). ThescFv construct was expressed in bacteria and purified using C-terminalpoly-histidine (HHHHHH) tag.

FIG. 27 shows the amino acid sequence of a MIN-E6 scFv (single chainfragment variable) design heavy chain variable (MIN-E6 VH7)—linker—lightchain variable). The scFv construct was expressed in bacteria andpurified using C-terminal poly-histidine (HHHHHH) tag.

FIG. 28 shows amino acid sequence for the 2D6C3 Kappa Chain VariableRegion. CDR1: RSSQTIVHSNGNTYLE (SEQ ID NO:70); CDR2: KVSNRFS (SEQ IDNO:71); and CDR3: FQGSHVPFT (SEQ ID NO:72).

FIG. 29 shows amino acid sequence for the 2D6C3 Heavy Chain VariableRegion. CDR1: GYAMS (SEQ ID NO:73); CDR2: TISSGGTYIYYPDSVKG (SEQ IDNO:74); and CDR3: LGGDNYYEY (SEQ ID NO:75).

FIG. 30 shows amino acid sequence for the 2D6C8 Kappa Chain VariableRegion. CDR1: RASKSVSTSGYSYMH (SEQ ID NO:76); CDR2: LVSNLES (SEQ IDNO:77); and CDR3: QHIRELTRSE (SEQ ID NO:78).

FIG. 31 shows amino acid sequence for the 2D6C8 Heavy Chain VariableRegion. CDR1: GYAMS (SEQ ID NO:79); CDR2: TISSGGTYIYYPDSVKG (SEQ IDNO:80); and CDR3: LGGDNYYEY (SEQ ID NO:81).

FIG. 32 shows amino acid sequence for the 3C2B1 Kappa Chain VariableRegion. CDR1: RASKSISTSDYNYIH (SEQ ID NO:82); CDR2: LASNLES (SEQ IDNO:83); and CDR3: QHSRELPLTF (SEQ ID NO:84).

FIG. 33 shows amino acid sequence for the 3C2B1 Heavy Chain VariableRegion. CDR1: TYTMS (SEQ ID NO:85); CDR2: TISTGGDKTYYSDSVKG (SEQ IDNO:86); and CDR3: GTTAMYYYAM (SEQ ID NO:87).

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses antibodies and antibody variants thatmodulate a pathway involving MUC1* wherein one set of antibodiespreferentially binds to MUC1* as it exists on stem cells but does notrecognize MUC1* on cancer cells as well and another set of antibodiesthat preferentially binds to MUC1* as it exists on cancer cells but doesnot recognize MUC1* on stem cells as well. The present invention furtherdiscloses methods for identifying other antibodies that fall into thesecategories. The invention further discloses methods for using the firstset of antibodies, hereafter referred to as “stem cell antibodies”, forstimulating stem cell growth in vitro and in vivo. The invention alsodiscloses methods for using the second set of antibodies, hereafterreferred to as “cancer cell antibodies”, for inhibiting cancer cellgrowth in vitro and in vivo.

In the present application, as well as in all of the applications fromwhich the present application claims priority, names that are given tothe antibodies that are disclosed in the priority applications as wellas in the present application are consistent. For instance, the cancerspecific antibodies MIN-C2 (also referred to herein as well as in theapplications from which the present application claims priority as “C2”)or MIN-E6 (also referred to herein as well as in the applications fromwhich the present application claims priority as “E6”) are the sameantibodies structurally and sequence-wise as referred to in the presentapplication as in the applications from which the present applicationclaims priority. Likewise, the stem cell specific antibodies 2D6C3 (alsoreferred to herein as well as in the applications from which the presentapplication claims priority as “C3”) or 2D6C8 (also referred to hereinas well as in the applications from which the present application claimspriority as “C8”) are the same antibodies structurally and sequence-wiseas referred to in the present application as in the applications fromwhich the present application claims priority.

In a preferred embodiment, a bivalent antibody selected from the set ofstem cell antibodies is administered to a patient for stimulating thegrowth of the patient's stem cells or progenitor cells. In a preferredembodiment, the progenitor cells are hematopoietic stem cells. Inanother embodiment, a bivalent antibody selected from the set of stemcell antibodies is used in vivo for stimulating the growth of apatient's stem or progenitor cells that have been mobilized usinganother agent, such as a CSF. In another embodiment, a bivalent antibodyselected from the set of stem cell antibodies is used in vitro forstimulating the growth of a person's mobilized stem cells that have beenextracted from the host for later transplantation either autologously oras donor cells transplanted into another allogeneically. In yet anotherembodiment, a bivalent antibody selected from the set of stem cellantibodies is used to stimulate the growth of stem cells in vivo. Inanother embodiment, a bivalent antibody selected from the set of stemcell antibodies is used for inducing pluripotency or inducing a lessdifferentiated state in a set of cells. In another embodiment,antibodies selected from the set of stem cell antibodies is used toidentify, select, isolate, or capture, including capture on a growthsurface, stem or progenitor cells. In a preferred embodiment the stemand/or progenitor cells referred to above are human in origin. Stem cellantibodies can be used to accelerate the recovery of blood cells fromeffects of exposure to radiation, toxins or chemotherapy, used afterbone marrow transplant, used before or after stem cell transplant whichmay be transplanted into the peripheral blood, and or to treat a patientwho could benefit from increased production of hematopoietic stem cellsor blood cells or their progenitors.

In one embodiment, antibodies selected from the set of cancer cellantibodies are used to treat cancer patients. In a preferred embodiment,antibodies selected from the set of cancer cell antibodies prevent thedimerization of the MUC1* portion of the MUC1 protein exemplified bymost of the PSMGFR sequence N-terminusGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA-C terminus (SEQ ID NO:1).In a more preferred embodiment, antibodies selected from the set ofcancer cell antibodies, used to treat a cancer patient, are monovalent,including Fabs, single chain constructs as well as other antibodiesincluding engineered antibody-like agents that bind to a portion of thePSMGFR sequence and prevent its ligand-induced dimerization.

MUC1* generally refers to a MUC1 protein or alternative splice isoformthat is devoid of some or all of its self-aggregation domainGFLGLSNIKFRPGSVVVQLTLAFREG (SEQ ID NO:2). In particular, MUC1* refers toMUC1 variants that lack the tandem repeat units. Most often MUC1* is atransmembrane cleavage product whose extra cellular domain consistsprimarily of a significant portion of the PSMGFR sequence. Because MUC1can be cleaved at a number of positions, its exact site of cleavage mayvary from one cell type to another.

In general, antibodies that belong to the group termed herein as “cancercell antibodies” bind to the 35 amino acids that are at the C-terminalend of the PSMGFR sequence: QFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGA (SEQ IDNO:3, also referred to in some figures as “N delta 10”).

In general, antibodies that belong to the group termed herein as “stemcell antibodies” bind to the 35 amino acids that are at the N-terminalend of the PSMGFR sequence: GTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV (SEQ IDNO:4, also referred to in some figures as “C delta 10”) but may alsobind a peptide extended N-terminally from the peptide of SEQ ID NO:4,namely including ten (10) amino acids N-terminal to SEQ ID NO:4, whichare VVQLTLAFRE (SEQ ID NO:5). Alternatively, antibodies that belong tothe group of stem cell antibodies are selected based on their ability tobind to the peptide of sequenceVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDV (SEQ ID NO:6). In apreferred embodiment, the stem cell antibodies do not bind to thepeptide SEQ ID NO:3.

Antibodies belonging to the set of cancer cell antibodies bind to apeptide containing 6-35 consecutive amino acids of the peptide of SEQ IDNO:3, which peptide may further contain up to 4 amino acidsubstitutions.

Antibodies belonging to the set of stem cell antibodies bind to apeptide containing 6-35 consecutive amino acids sequence of the peptideof SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, which peptide may furthercontain up to 4 amino acid substitutions.

To generate cancer cell antibodies, the immunogenic or antigenic peptideused contains 6-35 consecutive amino acids sequence of the peptide ofSEQ ID NO:3, which peptide may further contain up to 4 amino acidsubstitutions. In a preferred embodiment, the portions of the peptidethat are more C-terminal are used to generate cancer cell antibodies.For example peptides containing amino acids ASRYNLTISDVSVSDVPFPFSAQSGA(SEQ ID NO:7) or DVSVSDVPFPFSAQSGA (SEQ ID NO:8) are used to generatecancer cell antibodies. For the generation of cancer cell antibodies ofthe invention, the invention also includes methods for generatinghumanized antibodies, selecting human antibodies, or generatingantibody-like proteins in which the peptide is not used to immunize ananimal but rather is used, or the nucleic acids coding for it are used,to select an antibody or antibody-like protein that recognizes at least6 consecutive amino acids from the peptide of SEQ ID NO:3, such as thepeptide of SEQ ID NO:7 or SEQ ID NO:8. Further, the peptides of SEQ IDNO:3, SEQ ID NO:7 and SEQ ID NO:8 can be used to select for antibodiesthat specifically modulate the growth of cancer cells but not stem cellswith antibodies that bind to the more C-terminal portions such as thepeptide of SEQ ID NO:7, preferred wherein SEQ ID NO:8 is especiallypreferred. Phage display technology may be used to select antibodies orantibody-like proteins that bind to the peptides that identify it as acancer specific antibody. Alternatively, antibodies are generated usingthe entire PSMGFR peptide and cancer cell antibodies are selected basedon their ability to bind to peptides that contain 6-35 consecutive aminoacids sequence of the peptide of SEQ ID NO:3 or SEQ ID NO:7 or SEQ IDNO:8, which peptide may further contain up to 4 amino acidsubstitutions.

To generate stem cell antibodies, the immunogenic or antigenic peptideused contains 6-35 consecutive amino acids sequence of the peptide ofSEQ ID NO:4 or SEQ ID NO:5 or a peptide that has sequences from both, asin SEQ ID NO:6, which peptide may further contain up to 4 amino acidsubstitutions. In a preferred embodiment, the portions of the peptidethat are more N-terminal are used to generate stem cell antibodies. Forexample peptides containing amino acidsVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNL (SEQ ID NO:9) are preferred for thegeneration of stem cell antibodies. More preferred are peptides thatcontain consecutive amino acids from the peptide. Alternatively,antibodies are generated using the entire PSMGFR peptide and stem cellantibodies are selected based on their ability to bind to peptides thatcontain 6-35 consecutive amino acids sequence of the peptide of SEQ IDNO:4 or SEQ ID NO:5 or SEQ ID NO:6, which peptide may further contain upto 4 amino acid substitutions.

GTINVHDVETQFNQYKTEAASRYNL (SEQ ID NO:10) for the generation or selectionof stem cell specific antibodies. Still more preferred are peptidescontaining consecutive amino acids from GTINVHDVETQFNQY (SEQ ID NO:11).For the generation of stem cell antibodies, the invention also includesmethods for generating humanized antibodies, selecting human antibodies,or generating antibody-like proteins in which the peptide is not used toimmunize an animal but rather is used, or the nucleic acids coding forit are used, to select an antibody or antibody-like protein thatrecognizes at least 6 consecutive amino acids from the peptide of SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,wherein SEQ ID NO:10 is especially preferred. Phage display technologymay be used to select antibodies or antibody-like proteins that bind tothe peptides that identify it as a stem cell specific antibody.

In a preferred embodiment, antibodies belonging to the set of cancercell antibodies will bind to MUC1-positive cancer cells as determined byICC, FACS or other similar analysis including ELISAs and phage displayin which the antibody binds to a peptide containing some or all of theamino acids of SEQ ID NO:3. In a preferred embodiment, the antibodybinds to a peptide containing the last 20 amino acids at the C-terminusof SEQ ID NO:3. In a more preferred embodiment the antibody binds to apeptide containing only the last 10 amino acids at the C-terminus of thepeptide of SEQ ID NO:3. In a still more preferred embodiment, antibodiesbelonging to the set of cancer cell antibodies bind to MUC1-positivecancer cells but not to stem or progenitor cells. In addition,antibodies belonging to the cancer cell antibody set are selected byvirtue of their ability to stimulate cancer cell growth when they arebivalent and inhibit cancer cell growth when monovalent, e.g. Fab. In ayet more preferred embodiment neither the bivalent nor the monovalentform of the antibody will affect the growth of stem cells.

In a preferred embodiment, antibodies belonging to the set of stem cellantibodies bind to stem and/or progenitor cells as determined by ICC,FACS or other similar analysis including ELISAs and phage display inwhich the antibody binds to a peptide containing some or all of theamino acids of SEQ ID NO:4. In a preferred embodiment, the stem and/orprogenitor cells are human in origin. In another preferred embodiment,the antibody binds to a peptide containing only the 20 amino acids atthe N-terminus of SEQ ID NO:4. In a more preferred embodiment theantibody will bind to a peptide containing only the 10 amino acids atthe N-terminus of the peptide of SEQ ID NO:4. Alternatively, theantibody will bind to the peptide of SEQ ID NO:5, SEQ ID NO:6, or apeptide containing consecutive amino acids from the combined SEQ IDNOS:4 and 5. In a still more preferred embodiment, antibodies belongingto the set of stem cell antibodies will bind to human stem and/orprogenitor cells but not to MUC1-positive cancer cells. In addition,antibodies belonging to the stem cell antibody set will be selected byvirtue of their ability to stimulate stem and/or progenitor cell growthwhen they are bivalent and inhibit stem and/or progenitor cell growthwhen monovalent, e.g. Fab. In a yet more preferred embodiment neitherthe bivalent nor the monovalent form of the antibody will affect thegrowth of cancer cells.

Monoclonal antibodies were generated by standard methods andantibody-producing hybridomas were selected based upon the selectioncriteria set out herein. Monoclonal antibodies 2D6C3 and 2D6C8 (alsoreferred to herein as C3 and C8 respectively) were identified as stemcell antibodies. Their sequences are given in FIGS. 28 to 33. Monoclonalantibodies C2 and E6 were identified as cancer cell specific antibodies.The sequences of monoclonal antibodies C2 (MIN-C2) and E6 (MIN-E6) aregiven in FIGS. 24 to 27.

gaggtccagctggaggagtcagggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtagtggtggtacttatatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgggggggataattactacgaatacttcgatgtctggggcgcagggaccacggtcaccgtctcctccgccaaaacgacacccccatctgtctat (SEQ ID NO:12) describes MIN-C2 Heavychain variable region.

EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYI-DVWGAGTTVTVSSAKTTPPSVY (SEQ ID NO:13) describes MIN-C2 Heavy chainvariable region.

gacattgtgatcacacagtctacagcttccttaggtgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtaccaacagagaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgttcacgttcggaggggggaccaagctggagataaaacgggctgatgctgcaccaactgtatcc (SEQ ID NO:14) describes MIN-C2 Kappa chain variable region.

DIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGGTKLEIKRA DAAPTVS(SEQ ID NO:15) describes MIN-C2 Kappa chain variable region.

gaggttaagctggaggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtagatatggcatgtcttgggttcgccagactccagacaagaggctggagtgggtcgcaaccattagtagtggtggtacttacatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtattactgtgcaagggataactacggtagtagctacgactatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagccaaaacaacagccccatcggtctat (SEQ ID NO:16) describes MIN-E6Heavy chain-7 variable region.

EVKLEESGGDLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPDKRLEWVATISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDNYGSSYDYAMDYWGQGTSVTVSSAKTTAPSVY (SEQ ID NO:17) describes MIN-E6 Heavy chain-7variable region.

gaggtaaagctggaggagtctgggggagacttagtgaagcctggagggtccctgaaactctcctgtgtagtctctggattcactttcagtagatatggcatgtcttgggttcgccagactccaggcaagaggctggagtgggtcgcaaccattagtggtggcggtacttacatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaagtctgaggacacagccatgtatcactgtacaagggataactacggtaggaactacgactacggtatggactactggggtcaaggaacctcagtcaccgtctcctcagccaaaacaacagccccatcggtctatccactggcccctgtgtgtggagatacaactggctcctcggtgactctaggatgcctggtcaag (SEQ ID NO:18) describes MIN-E6 Heavy chain-8 variable region.

EVKLEESGGDLVKPGGSLKLSCVVSGFTFSRYGMSWVRQTPGKRLEWVATISGGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYHCTRDNYGRNYDYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVK (SEQ ID NO:19) describesMIN-E6 Heavy chain-8 variable region.

gatattgtgatcacccagactacagcaatcatgtctgcatctccaggggaggaggtcaccctaacctgcagtgccacctcaagtgtaagttacatacactggttccagcagaggccaggcacttctcccaaactctggatttatagcacatccaacctggcttctggagtccctgttcgcttcagtggcagtggatatgggacctcttactctctcacaatcagccgaatggaggctgaagatgctgccacttattactgccagcaaaggagtagttccccattcacgttcggctcggggacaaagttggaaataaaacgggctgatgctgcaccaactgtatcc(SEQ ID NO:20) describes MIN-E6 Kappa chain variable region.

DIVITQTTAIMSASPGEEVTLTCSATSSVSYIHWFQQRPGTSPKLWIYSTSNLASGVPVRFSGSGYGTSYSLTISRMEAEDAATYYCQQRSSSPFTFGSGTKLEIKRADAAPTVS (SEQ IDNO:21) describes MIN-E6 Kappa chain variable region.

gaggtccagctggaggagtctgggggaggcttagtgaagcctggagggtccctgaaactctcctgtgcagcctctggattcactttcagtggctatgccatgtcttgggttcgccagactccggagaagaggctggagtgggtcgcaaccattagtagtggtggtacttatatctactatccagacagtgtgaaggggcgattcaccatctccagagacaatgccaagaacaccctgtacctgcaaatgagcagtctgaggtctgaggacacggccatgtattactgtgcaagacttgggggggataattactacgaatacttcgatgtctggggcgcagggaccacggtcaccgtctcctccgccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccagccagcaggaccgcg (SEQ ID NO:22) describes MIN-C2 Fab Heavy chain.

gacattgtgatcacacagtctacagcttccttaggtgtatctctggggcagagggccaccatctcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtaccaacagagaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgttcacgttcggaggggggaccaagctggagataaaacgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgt (SEQ ID NO:23) describes MIN-C2 Fab Kappa chain.

EVQLEESGGGLVKPGGSLKLSCAASGFTFSGYAMSWVRQTPEKRLEWVATISSGGTYIYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARLGGDNYYEYFDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASRTA (SEQ ID NO:24) describesMIN-C2 Fab Heavy chain.

DIVITQSTASLGVSLGQRATISCRASKSVSTSGYSYMHWYQQRPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPFTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:25) describesMIN-C2 Fab Kappa chain.

RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO:26)describes MIN-C2 light CL region amino acid sequence.

FDVWGAGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASRTA (SEQ ID NO:27)describes MIN-C2 heavy chain CH1 region amino acid sequence.

DIVITQSTASLGVSLGQRATISC (SEQ ID NO:28) describes MIN-C2 light chainvariable framework region 1 (FWR1) amino acid sequence.

DIVITQTTAIMSASPGEEVTLTC (SEQ ID NO:29) describes MIN-E6 light chainvariable framework region 1 (FWR1) amino acid sequence.

RASKSVSTSGYSYMH (SEQ ID NO:30) describes MIN-C2 light chain variablecomplementarity determining region 1 (CDR1) amino acid sequence.

SATSSVSYIH (SEQ ID NO:31) describes MIN-E6 light chain variablecomplementarity determining region 1 (CDR1) amino acid sequence.

WYQQRPGQPPKLLIY (SEQ ID NO:32) describes MIN-C2 light chain variableframework region 2 (FWR2) amino acid sequence.

WFQQRPGTSPKLWIY (SEQ ID NO:33) describes MIN-E6 light chain variableframework region 2 (FWR2) amino acid sequence.

LASNLES (SEQ ID NO:34) describes MIN-C2 light chain variablecomplementarity determining region 2 (CDR2) amino acid sequence.

STSNLAS (SEQ ID NO:35) describes MIN-E6 light chain variablecomplementarity determining region 2 (CDR2) amino acid sequence.

GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC (SEQ ID NO:36) describes MIN-C2 lightchain variable framework region 3 (FWR3) amino acid sequence.

GVPVRFSGSGYGTSYSLTISRMEAEDAATYYC (SEQ ID NO:37) describes MIN-E6 lightchain variable framework region 3 (FWR3) amino acid sequence.

QHSRELPFT (SEQ ID NO:38) describes MIN-C2 light chain variablecomplementarity determining region 3 (CDR3) amino acid sequence.

QQRSSSPFT (SEQ ID NO:39) describes MIN-E6 light chain variablecomplementarity determining region 3 (CDR3) amino acid sequence.

EVQLEESGGGLVKPGGSLKLSCAASGFTFS (SEQ ID NO:40) describes MIN-C2 heavychain variable framework region 1 (FWR1) amino acid sequence.

EVKLEESGGDLVKPGGSLKLSCAASGFTFS (SEQ ID NO:41) describes MIN-E6-7 heavychain variable framework region 1 (FWR1) amino acid sequence.

EVKLEESGGDLVKPGGSLKLSCVVSGFTFS (SEQ ID NO:42) describes MIN-E6-8 heavychain variable framework region 1 (FWR1) amino acid sequence.

GYAMS (SEQ ID NO:43) describes MIN-C2 heavy chain variablecomplementarity determining region 1 (CDR1) amino acid sequence.

RYGMS (SEQ ID NO:44) describes MIN-E6-7 heavy chain variablecomplementarity determining region 1 (CDR1) amino acid sequence.

RYGMS (SEQ ID NO:45) describes MIN-E6-8 heavy chain variablecomplementarity determining region 1 (CDR1) amino acid sequence.

WVRQTPEKRLEWVA (SEQ ID NO:46) describes MIN-C2 heavy chain variableframework region 2 (FWR2) amino acid sequence.

WVRQTPDKRLEWVA (SEQ ID NO:47) describes MIN-E6-7 heavy chain variableframework region 2 (FWR2) amino acid sequence.

WVRQTPGKRLEWVA (SEQ ID NO:48) describes MIN-E6-8 heavy chain variableframework region 2 (FWR2) amino acid sequence.

TISSGGTYIYYPDSVKG (SEQ ID NO:49) describes MIN-C2 heavy chain variablecomplementarity determining region 2 (CDR2) amino acid sequence.

TISSGGTYIYYPDSVKG (SEQ ID NO:50) describes MIN-E6-7 heavy chain variablecomplementarity determining region 2 (CDR2) amino acid sequence.

TISGGGTYIYYPDSVKG (SEQ ID NO:51) describes MIN-E6-8 heavy chain variablecomplementarity determining region 2 (CDR2) amino acid sequence.

RFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR (SEQ ID NO:52) describes MIN-C2 heavychain variable framework region 3 (FWR3) amino acid sequence.

RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR (SEQ ID NO:53) describes MIN-E6-7 heavychain variable framework region 3 (FWR3) amino acid sequence.

RFTISRDNAKNTLYLQMSSLKSEDTAMYHCTR (SEQ ID NO:54) describes MIN-E6-8 heavychain variable framework region 3 (FWR3) amino acid sequence.

LGGDNYYEY (SEQ ID NO:55) describes MIN-C2 heavy chain variablecomplementarity determining region 3 (CDR3) amino acid sequence.

DNYGSSYDYA (SEQ ID NO:56) describes MIN-E6-7 heavy chain variablecomplementarity determining region 3 (CDR3) amino acid sequence.

DNYGRNYDYG (SEQ ID NO:57) describes MIN-E6-8 heavy chain variablecomplementarity determining region 3 (CDR3) amino acid sequence.

EVQLVESGGGLVKPGGSLRLSCA ASGFTFS (SEQ ID NO:58) describes IGHV3 (namefrom Igblast): FWR1: Human IgG antibody framework region sequence with84.7% homology (249/294) to variable heavy chain region of MIN-C2.

WVRQAPGKGLEWVS (SEQ ID NO:59) describes IGHV3 (name from Igblast): FWR2:Human IgG antibody framework region sequence with 84.7% homology(249/294) to variable heavy chain region of MIN-C2.

RFTISRDNAKNSLYLQMNSLRAEDTAV (SEQ ID NO:60) describes IGHV3 (name fromIgblast): FWR3: Human IgG antibody framework region sequence with 84.7%homology (249/294) to variable heavy chain region of MIN-C2.

DIVLTQSPASLAVSPGQRATITC (SEQ ID NO:61) describes IGkV7 (name fromIgblast): FWR1: Human IgG antibody framework region sequence with 76.4%homology (226/296) to variable light chain region of MIN-C2.

WYQQKPGQPPKLLIY (SEQ ID NO:62) describes IGkV7 (name from Igblast):FWR2: Human IgG antibody framework region sequence with 76.4% homology(226/296) to variable light chain region of MIN-C2.

GVPARFSGSGSGTDFTLTINPVEANDTANYY (SEQ ID NO:63) describes IGkV7 (namefrom Igblast): FWR 3: Human IgG antibody framework region sequence with76.4% homology (226/296) to variable light chain region of MIN-C2.

EVQLVESGGGLVKPGGSLRLSCAASGFTFS (SEQ ID NO:64) describes IGHV3 (name fromIgblast): FWR1: Human IgG antibody framework region sequence with 84.1%homology (249/296) to variable heavy chain region of MIN-E6.

WVRQAPGKGLEWVS (SEQ ID NO:65) describes IGHV3 (name from Igblast): FWR2:Human IgG antibody framework region sequence with 84.1% homology(249/296) to variable heavy chain region of MIN-E6.

RFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR (SEQ ID NO:66) describes IGHV3 (namefrom Igblast): FWR3: Human IgG antibody framework region sequence with84.1% homology (249/296) to variable heavy chain region of MIN-E6.

EIVMTQSPATLSVSPGERATLSC (SEQ ID NO:67) describes IGkV3 (name fromIgblast): FWR1: Human IgG antibody framework region sequence with 69.5%homology (187/269) to variable light chain region of MIN-E6.

WFQQRPGTSPK LLIY (SEQ ID NO:68) describes IGkV3 (name from Igblast):FWR2: Human IgG antibody framework region sequence with 69.5% homology(187/269) to variable light chain region of MIN-E6.

GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC (SEQ ID NO:69) describes IGkV3 (namefrom Igblast): FWR3: Human IgG antibody framework region sequence with69.5% homology (187/269) to variable light chain region of MIN-E6.

Antibodies produced by the various clones were tested first by ELISA.Ideally, cancer cell specific antibodies would bind to the peptide SEQID NO:3 lacking the last 10 N-terminal amino acids of the PSMGFR peptide(SEQ ID NO:1), but not to the peptide of SEQ ID NO:4, lacking the last10 amino acids at the C-terminus of the PSMGFR peptide. Ideally, thestem cell specific antibodies would have the opposite binding pattern;they should bind to peptide of SEQ ID NO:4 but not to peptide of SEQ IDNO:3. By ELISA, monoclonal antibodies 2D6C3 and 2D6C8 (also referred toherein as C3 and C8 respectively) bound to the peptide of SEQ ID NO:4but not to the peptide of SEQ ID NO:3. Conversely, monoclonal antibodiesC2 and E6 bound to the peptide of SEQ ID NO:3 but not to the peptide ofSEQ ID NO:4, see FIG. 2.

In another method to identify monoclonal antibodies that were specificfor stem cells, antibodies were coated onto the surface of a cellculture plate. Because human stem cells are not adherent, they shouldnot attach to the plate unless the plate is coated with an antibody thatrecognizes a receptor on the surface of the stem cells. In this way,clones 2D6C3 and 2D6C8 (also referred to herein as C3 and C8respectively) were identified as being stem cell specific antibodies. Ina preferred embodiment, the antibodies bound to a peptide whose sequencecorresponds to the MUC1* extra cellular domain but not to the membraneproximal portion (SEQ ID NO:3). In a more preferred embodiment, theantibodies bound to the distal portion peptide (SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6) of the PSMGFR peptide and not to the membraneproximal portion (SEQ ID NO:3),

The monoclonal antibodies can additionally be tested for bindingspecificity by FACS to determine if they bound to cancer cells or stemcells. FIGS. 3-6 show FACS data that collectively show that the cancerspecific antibodies C2 (MIN-C2) and E6 (MIN-E6) only bind toMUC1-positive cancer cells but not to human stem cells. Conversely,FIGS. 3-6 show that stem cell antibodies 2D6C3 and 2D6C8 only bind tostem cells and not to cancer cells.

As yet another test to determine the specificity of monoclonalantibodies, their effect on the growth of either stem cells or cancercells can be tested. As can be seen in the cartoon of FIG. 1, bivalentanti-MUC1* antibodies dimerize the extra cellular domain of MUC1* andstimulate growth, while the monovalent Fabs inhibit growth. The bivalentforms of the antibodies were tested for their ability to stimulate thegrowth of human stem cells and the monovalent Fabs were tested for theirability to inhibit the growth of the stem cells. FIGS. 7-9 showphotographs and plotted data showing that bivalent stem cell antibodies2D6C3 and 2D6C8 (C3 and C8) stimulate human stem cell growth. FIGS.10-12 show that the Fab of the stem cell antibody C3 at 100 uM inhibitsgrowth of stem cells but the Fabs of the cancer specific antibodies C2(MIN-C2) and E6 (MIN-E6) do not. FIGS. 13-15 show that the Fabs of thecancer specific antibodies C2 (MIN-C2) and E6 (MIN-E6) inhibit thegrowth of MUC1-positive cancer cells but the Fabs of the stem cellspecific antibodies 2D6C3 and 2D6C8 (C3 and C8) do not. FIG. 16 showsthat none of the Fabs of the cancer specific or stem specific antibodieshave any effect on MUC1-negative cells, such as PC3 prostate cancercells.

As yet a further test of binding specificity, antibodies selected to bestem specific can be tested in vivo and assayed for their ability tostimulate stem or progenitor cell growth. Cancer specific antibodies canbe tested in vivo for their ability to inhibit cancer cell growth. Inone example, to test the in vivo effects of a cancer cell antibodyidentified using methods of the invention, female nude mice, into whichestrogen pellets had been implanted, were xenografted with human breasttumor cells (T47D). Tumors were allowed to engraft for 14 days beforethe mice were treated with 80 mg/kg of the E6 Fab 2-times per week. Anequal number of control mice were injected with buffer alone. Treatmentwas suspended during the 14 day period between Day 31 and Day 45 todetermine whether the treated mice would develop a resistance to thetreatment. The graph of FIG. 17 shows that the E6 Fab effectivelyreduced tumor growth rate and volume compared to the control mice. Thefact that after a 14 day lapse of treatment, the mice continued torespond to the E6 Fab shows that they did not develop a resistance tothe drug. FIG. 18 shows photographs of two of the mice from the study.

In another in vivo study, male NOD/SCID mice were xenografted with humanprostate tumors (DU-145). Tumors were allowed to develop for 14 daysbefore treatment or mock treatment was begun. 60 days after tumorimplantation the groups were switched; the mice receiving the cancercell antibody E6 Fab were then given the buffer alone and the controlgroup began receiving the E6 Fab. Treatment was with 160 mg/kg of the E6Fab given every 24 hours. The graph of FIG. 19 shows that the treatedgroup had significantly smaller tumors than the control group. Beforethe treatment groups were swapped, the tumors in the control group weremore than 3-times larger than the treated group. In addition, only oneof the treated mice had an increase in tumor growth when treatment washalted. FIG. 20 is a Western blot for MUC1* that shows that even after 2weeks without treatment, the treated group on average had less MUC1*than the control group. Additionally, the graph of FIG. 20 shows thatthe treated group had an increase in the amount of microRNA-145(miR-145) that they produced, which indicates that the E6 Fab treatmentcaused the cancer cells to differentiate which limited their ability toself-replicate.

In another animal study to test the efficacy of the cancer cellantibodies identified using methods of the invention, male NOD/SCID micewere xenografted with human prostate tumors (DU-145). Tumors wereallowed to develop for 27 days before treatment or mock treatment wasbegun. The animals were divided into four groups wherein groups 1 and 2had tumor volumes 350-500 mm³ and groups 3 and 4 had tumor volumes175-300 mm³. Treatment was 160 mg/kg of the E6 Fab given every 24 hours(groups 1 and 3) and the mock treatment was buffer alone (groups 2 and4). The graph of FIG. 21 shows that the tumors in the control mice grewat the same rate regardless of their starting volume. Although bothtreated groups had tumors that were much smaller than the controlgroups, mice whose tumors were in the 175-300 mm³ responded better thanthe groups bearing tumors 350-500 mm³ before treatment began. FIG. 22shows representative photographs of the mice from the control group andFIG. 23 shows representative photographs of the mice from the treatedgroup.

Thus, stem cell specific antibodies 2D6C3 and 2D6C8 (C3 and C8), as wellas other monoclonal, polyclonals and antibody variants identified orselected using methods of the invention can be used much in the same waythat ESAs and CSFs like Epogen and Leukine are used, including toaccelerate the recovery of blood cells from effects of chemotherapy,used after bone marrow transplant, used before or after stem celltransplant which may be transplanted into the peripheral blood, and orto treat a patient who could benefit from increased production ofhematopoietic stem cells or blood cells or their progenitors. There areseveral methods for humanizing stem cell specific antibodies 2D6C3 and2D6C8 (C3 and C8), as well as other monoclonal, polyclonals and antibodyvariants identified or selected using methods of the invention, whereinconstant regions of the mouse monoclonal antibodies are replaced withhomologous human constant regions and wherein the variable region may beleft as the mouse sequences or replaced by homologous human sequences.Phage display techniques can be used along with peptides of theinvention to select the antibodies that bind best to the preferred MUC1*peptide. Alternatively, fully human antibody libraries can be screenedde novo for their ability to bind to either the peptides used forselection of stem specific antibodies or the peptides used for selectionof the cancer specific antibodies. If a stem specific antibody isdesired, candidate antibodies can be screened for their ability to bindto cancer specific peptides in a de-selection process.

Cancer specific antibodies such as C2 (MIN-C2) and E6 (MIN-E6) as wellas other monoclonal, polyclonals and antibody variants identified orselected using methods of the invention can be used to treat cancerpatients with reduced risk of killing the patient's stem cells or bloodcell precursors and progenitors. There are several methods forhumanizing cancer specific antibodies C2 (MIN-C2) and E6 (MIN-E6), aswell as other monoclonal, polyclonals and antibody variants identifiedor selected using methods of the invention, wherein constant regions ofthe mouse monoclonal antibodies are replaced with homologous humanconstant regions and wherein the variable region may be left as themouse sequences or replaced by homologous human sequences. Phage displaytechniques can be used along with peptides of the invention to selectthe antibodies that bind best to the preferred MUC1* peptide.Alternatively, fully human antibody libraries can be screened de novofor their ability to bind to either the peptides used for selection ofstem specific antibodies or the peptides used for selection of thecancer specific antibodies. If a cancer specific antibody is desired,candidate antibodies can be screened for their ability to bind to stemspecific peptides in a de-selection process.

The invention includes antibodies as well as antibody-like proteins,including but not limited to polyclonal, monoclonal, chimeras,humanized, single chain, antibody fragments and the like. In addition,the invention includes the use of protein scaffolds for generatingantibody mimics to obtain proteins that can be characterized by bindingassays described herein and according to methods of the invention asbeing stem cell antibodies or cancer cell antibodies and thus being ableto bind specifically to either MUC1* as it exists on cancer cells, orbind to MUC1* as it exists on stem cells, progenitor cells or engineeredcells that express a form of MUC1*. The invention further includes usingmethods set forth here to identify antibodies that recognize specificepitopes, within the MUC1* extra cellular domain, that are expressed ondifferent types of progenitor cells or non-tumor cancer cells.

In general, antibodies for the treatment of cancer should inhibitdimerization of MUC1* receptor. Thus in a preferred embodiment,antibodies for the treatment of MUC1-positive cancers are monovalent,such as Fabs, single chain antibodies, or bispecific antibodies. Theinvention does contemplate the use of antibodies such as pentavalentIgMs for the treatment of cancers because the multivalency acts tore-cluster the MUC1* extra cellular domain. However, bivalent antibodiesat high enough concentration bind one antibody to each receptor ratherthan one antibody per each one receptor and in so doing are alsoinhibitory to cancer growth and development.

In general, antibodies of the invention that are suitable forstimulating stem and progenitor cell growth are bivalent such that theyactivate growth, survival and pluripotency pathways by dimerizing theMUC1* extra cellular domain Stem and progenitor cell specific antibodiescan be used to treat patient suffering from anemia, low white blood cellcount, low platelets or any blood cell or blood progenitor celldeficiency. Additionally, the invention contemplates the use of stem andprogenitor specific antibodies for use as anti-aging therapeutics,agents to promote overall health, agents to enhance stem or progenitorcell growth or to enhance engraftment of cells. Stem and progenitorspecific antibodies described here may be administered to the patientsystemically or locally, as an injection or as a topical treatment, asin a medicament for the eye or other areas of the body that aremechanically accessible.

Bivalent stem cell specific antibodies 2D6C3 and 2D6C8 (C3 and C8), aswell as other monoclonal, polyclonals and antibody variants identifiedor selected using methods of the invention, can be used to stimulatestem cell growth in vitro or in vivo or can alternatively be used toenhance engraftment of stem cell transplants, irrespective of thesource. In another embodiment, stem cell specific bivalent antibodies ofthe invention are used to stimulate the growth of hematopoietic stemcells in vitro or in vivo. In some cases stem cell antibodies are usedto treat patients suffering from or at risk of developing anemia or lowwhite blood count. These patients may be suffering from cancer and maybe simultaneously treated with a cancer cell antibody of the inventionfor inhibiting their cancer cell growth.

In another aspect, antibodies and peptides of the invention are used fordiagnosis. Contacting a patient's cells with a cancer specific antibodyand getting significant binding would indicate that the patient hascancer. Similarly, contacting a patient's cells with a stem specificantibody and getting significant binding would indicate that the patientdoes not have cancer and would further identify those cells as stem orprogenitor cells. A patient's cells can be contacted by both types ofantibodies in order to distinguish stem cells from cancer cells. Suchdiagnosis can be carried out in vitro or in vivo. In vitro, a tissuespecimen, blood sample, or bodily fluid sample can be analyzed usingcancer or stem specific antibodies. In vivo, imaging agents can beattached to the antibodies of the invention to enable identification ofcancer or stem cells in a patient.

EXAMPLES Example 1 Development of Monoclonal Antibodies, 2D6C8 and 2D6C3(Also Referred to Here as C3 and C8) that Facilitate Human Stem CellAttachment to Surfaces

MUC1* monoclonal antibodies were identified that preferentially bound tothe portion of the MUC1* extra cellular domain that is more distal fromthe cell surface and these monoclonals were shown to better facilitatethe attachment of human ES and iPS cells to surfaces. Mice wereimmunized with a peptide that is defined by the PSMGFR sequence.Supernatants of hybridoma clones were tested by ELISA for their abilityto bind to the PSMGFR peptide and by FACS to determine which bound tolive, MUC1* positive cells. Hybridomas were further selected if theypreferentially bound to the PSMGFR peptide lacking 10 C-terminal aminoacids, but did not bind if the peptide lacked the 10 N-terminalpeptides. In addition, hybridomas were screened for their ability tofacilitate stem cell attachment to a surface such as a plastic cellculture plate. Of these clones two, 2D6C8 and 2D6C3 were selected thatwhen coated onto a surface captured stem cells and facilitated theirgrowth.

1.-18. (canceled)
 19. An antibody that specifically binds to an epitopeof the MUC1 protein expressed on human undifferentiated stem cells. 20.The antibody of claim 19, which specifically binds to at least sixconsecutive amino acids of the peptide of SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.
 21. The antibody ofclaim 19, which does not bind or binds significantly less to the peptideof SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:8.
 22. The antibody of claim19, which specifically binds to the peptide of SEQ ID NO:3 and does notbind to or binds significantly less to the peptide of SEQ ID NO:4. 23.The antibody of claim 19, which specifically binds to at least sixconsecutive amino acids of the peptide of SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, and does not bind toor binds significantly less to the peptide of SEQ ID NO:3, SEQ ID NO:7or SEQ ID NO:8.
 24. A method for treating a patient suffering fromcancer, comprising administering to a patient with MUC1-positive cancercells an antibody that specifically binds to an epitope of the MUC1protein that is expressed on cancer cells but is not expressed on humanundifferentiated stem cells, comprising obtaining an antibody thatspecifically binds to cancer cells, but not to stem cells comprisingselecting antibodies that bind to at least six consecutive amino acidsof a peptide of SEQ ID NO:3, SEQ ID NO:7 or SEQ ID NO:8, but do not bindor bind significantly less to at least six consecutive amino acids ofthe peptide of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, SEQID NO:10 or SEQ ID NO:11, wherein the antibody that specifically bindsto cancer cells, but not to stem cells is obtained; and administeringthe obtained antibody to the patient suffering from MUC-1 positivecancer.
 25. The method according to claim 24, wherein the antibody ismonovalent.
 26. The method according to claim 25, wherein the antibodyis scFv or Fab.
 27. The method according to claim 24, wherein theantibody is humanized.
 28. The method according to claim 24, wherein thepatient suffers from cancer and is receiving chemotherapy or radiation.29. The method according to claim 24, wherein the patient suffers fromchronic kidney disease.
 30. The method according to claim 24, whereinthe antibody specifically binds to at least six consecutive amino acidsof the peptide of SEQ ID NO:3.
 31. The method according to claim 24,wherein the antibody specifically binds to at least six consecutiveamino acids of the peptide of SEQ ID NO:7.
 32. The method according toclaim 24, wherein the antibody specifically binds to at least sixconsecutive amino acids of the peptide of SEQ ID NO:8.
 33. The methodaccording to claim 30, wherein the antibody does not bind or bindssignificantly less to at least six consecutive amino acids of thepeptide of SEQ ID NO:4.
 34. A method for treating a patient sufferingfrom cancer, comprising administering to a patient with MUC1-positivecancer cells an antibody that specifically binds to an epitope of theMUC1 protein that is expressed on cancer cells but is not expressed onhuman undifferentiated stem cells, comprising obtaining an antibody thatspecifically binds to cancer cells, but not to stem cells comprising (i)generating a mixed set of antibodies that recognize a peptide whosesequence is that of any of the peptides having sequence of SEQ IDNOS:1-11; and (ii) selecting antibodies that bind to at least sixconsecutive amino acids of a peptide of SEQ ID NO:3, SEQ ID NO:7 or SEQID NO:8, but do not bind or bind significantly less to at least sixconsecutive amino acids of the peptide of SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11, wherein the antibodythat specifically binds to cancer cells, but not to stem cells isobtained; and administering the obtained antibody to the patientsuffering from MUC-1 positive cancer.